Activated T Cells to Anergy Induction Differences Between Responses Of

Differences Between Responses of Naive and Activated T Cells to Anergy Induction Robert J. Hayashi, Dennis Y. Loh, Osami Kanagawa and Fanping Wang This information is current as of September 25, 2021. J Immunol 1998; 160:33-38; ; http://www.jimmunol.org/content/160/1/33

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Differences Between Responses of Naive and Activated T Cells to Anergy Induction1

Robert J. Hayashi,2* Dennis Y. Loh,† Osami Kanagawa,‡ and Fanping Wang†‡

T cell unresponsiveness to Ag stimulation can be induced by several means. The precise mechanism by which this process occurs remains poorly understood. Preincubating T cells with either EDCI-fixed APC or ionomycin is a proven means of inducing T cell anergy with reduced IL-2 production in response to Ag stimulation. Using T cells from mice expressing the TCR transgene DO11.10, which is specific for a peptide (323–339) derived from hen egg OVA, we demonstrate that naive cells obtained directly from the host are resistant to the anergy induction by either fixed APC or ionomycin. TCR transgenic mice also deficient -in the recombination-activating gene-2 (RAG-2؊/؊), preventing the formation of T cells with endogenous TCRs, were immu nized with OVA, and in vivo activated T cells with low expression of CD62 were isolated. These primed cells possess the same sensitivity to ionomycin-induced anergy as in vitro activated cell lines. This unresponsive state most profoundly affects Ag- Downloaded from induced IL-2 production, with IFN-␥ and IL-3 affected to a lesser degree and no effect observed on IL-4 production. Thus, T cells in vivo can be distinguished phenotypically by their susceptibility to anergic stimuli. Anergy so induced affects selected T cell functions. The Journal of Immunology, 1998, 160: 33–38.

g stimulation of T cells using chemically ﬁxed APC ren- erating this same anergic state in freshly isolated, naive T cells.

ders them unresponsive to subsequent rechallenge with Anergy induction has been shown to require an initial proliferative http://www.jimmunol.org/ A functional APC (1). Stimulation by an alternative response that transforms a naive T cell to a preactivated in vitro means, such as using Abs against the TCR complex or class II memory cell (14–16). Previous studies have attempted to demon- molecules in planar lipid membranes, generates similar anergic strate that freshly isolated T cells can be rendered anergic. How- states (2, 3). Whether the same pathway is used in each of these ever, most studies have incubated these cells several days in cul- modes is unclear, but such experimental models have indicated ture, which may, in fact, change their phenotype (17–19). Indeed, that a second signal in addition to TCR engagement is required for such cells have been shown to possess a phenotype of an activated activation and avoidance of anergy induction (4). More recent state, with low expression of CD62 (MEL-14) (20). Unfortunately, studies have suggested that B7-1 or B7-2 binding to CD28 may the analysis of Ag responses in naive, freshly isolated populations by guest on September 25, 2021 account for this second signal, and that stimulation of the TCR and is limited by the low frequency of T cells specific for any one engagement of the CD28 molecule are sufficient for T cell activa- particular Ag. Ideally, such studies should be performed analyzing tion and avoidance of the unresponsive state (5–7). a cell population with one receptor specificity. This would allow Subsequent studies analyzing the biochemical events in T cells exposed to anergic stimuli have suggested that these unresponsive for a specific Ag response to be evaluated after challenges with cells maintain persistently high intracellular Ca2ϩ levels (8). Fur- various stimuli. thermore, these investigations have demonstrated that increasing We have proceeded with such studies in mice that possess a the intracellular Ca2ϩ concentration with ionophores such as iono- transgene-derived Ag-negative TCR. This allows for the identifi- mycin is sufficient to establish the unresponsive state generated by cation and isolation of a specific T cell population in which the other anergic stimuli. Other biochemical and molecular events that response to a specific Ag can be monitored. The DO11.10, mouse have been observed in these cells include altered tyrosine phos- whose transgenic receptor is specific for a single polypeptide phorylation patterns of intracellular proteins (9, 10), defective ac- (323–339) contained within hen egg OVA presented by I-Ad class tivation of the Ras pathway (11, 12), and altered trans-activating II MHC molecules, was used for these studies (21). To ensure that activity of IL-2-specific proteins, such as activating protein-1 (13). cells with alternative TCRs would not affect these results, the T cell clones have primarily been used in these cited studies and transgene was introduced into a mouse line deficient in the recom- are thus cells that have been preactivated with Ag in vitro and bination-activating gene-2 (RAG-2)3 gene (22), preventing the for- maintained in culture. Little is known regarding the ability of gen- mation of T cells with endogenous TCRs. Using these mice, we have demonstrated that 1) naive T cells are resistant to anergy induction; 2) the in vivo activated T cell is also susceptible to Departments of *Pediatrics, †Medicine, and ‡Pathology, Washington University School of Medicine, St. Louis, MO 63110 anergy induction, indicating that this phenotypic change is not due Received for publication June 17, 1997. Accepted for publication September to the in vitro culture conditions; and 3) ionomycin-induced un- 12, 1997. responsiveness affects the production of IL-2, but other cytokines The costs of publication of this article were defrayed in part by the payment of expressed, such as Ag-induced IL-3 and IFN-␥, are affected to a page charges. This article must therefore be hereby marked advertisement in lesser degree, while IL-4 production remains unaffected. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institute of Health Grants K11AI01108 and RO1-AI30803. 2 Address correspondence and reprint requests to Dr. Robert J. Hayashi, Depart- ment of Pediatrics, Washington University School of Medicine, Box 8116, 660 3 Abbreviations used in this paper: RAG-2, recombination-activating gene-2; South Euclid, St. Louis, MO 63110. PKC, protein kinase C.

Materials and Methods Results Mice Naive T cells are resistant to anergy induction by EDCI-fixed The DO11.10 TCR transgenic mice were described previously (21) and APC bred at the Washington University School of Medicine (St. Louis, MO) in Treatment of T cell clones with stimuli such as anti-CD3 Abs or a pathogen-free facility with monitored surveillance and serologic testing Ϫ Ϫ Ag presented by chemically fixed APC results in the generation of of sentinel mice every 3 mo. The RAG-2 / mice were a gift from Dr. Y. Shinkai (22) and were used to establish DO11.10 TCR RAG-2Ϫ/Ϫ, H-2d an anergic state. T cells in this state are not capable of producing mice. DO11.10 TCR RAG-2Ϫ/Ϫ mice were identified as those mice pos- IL-2 upon challenge with Ag and functional APC. The effects of sessing T cells bearing the transgene-derived receptor using the Ab KJ1-26 such treatments on freshly isolated naive T cells have not been and lacking cells expressing B cell surface markers as determined by FACS clearly demonstrated. To facilitate this analysis, we used T cells analysis of peripheral blood. Male and female mice used in the experiments from mice possessing a transgene-derived TCR (DO11.10) that is discussed below were between 6 and 8 wk of age. d specific for OVA323–339 peptide presented by I-A . Most of this Flow cytometry mouse line’s peripheral T cells possess the transgenic TCR, pro- viding a naive T cell population whose function can be analyzed. Cell surface immunofluorescence analysis of T cell populations was per- Freshly isolated, naive T cells from DO11.10 transgenic mice and formed as described previously (10). In brief, 1 ϫ 106 cells were incubated with the Ab at saturating concentrations at 4°C for 30 min, washed, and cell lines generated from in vitro activated T cells from this same further incubated with FITC-, phycoerythrin-, or tricolor-conjugated ap- mouse strain were incubated with fixed APC plus peptide for 16 to propriate secondary reagents for an additional 30 min. Control samples 20 h. The cells were washed and rested for 3 days. Surviving T

were prepared in the same manner, but without primary Ab or with an cells were counted and stimulated with Ag and APC. The super- Downloaded from isotype-matched control. Samples were analyzed by a FACScan analyzer (Becton Dickinson, Mountain View, CA) using the CellQuest program. natants derived from these cultures were then assayed for IL-2 The mAbs used include KJ1-26, specific for an Id expressed on the trans- production. Surprisingly, naive T cells treated with fixed APC gene-derived TCR; DO11.10 (21); and MEL-14 (anti-CD62, PharMingen, were capable of producing amounts of IL-2 comparable to those San Diego, CA). Lymph node cells from OVA-primed mice were stained produced by untreated T cells (Fig. 1A). In contrast, in vitro cul- with KJ1-26 and biotinylated MEL-14, with a goat anti-mouse FITC sec- tured cell lines became anergic with exposure to Ag presented by ondary Ab (Caltag, San Francisco, CA) for KJ1-26, and with phycoerythrin-avidin secondary staining for MEL-14. CD62 low KL1-26-posi- fixed APC, with a failure to produce IL-2 upon rechallenge with http://www.jimmunol.org/ tive cells were separated by FACSVantge (Becton Dickinson). The Ag and functional APC. This experiment was performed three resulting populations were Ͼ98% pure. times, and in each instance, IL-2 production, in response to Ag stimulation, was maintained in the naive T cell population, while In vitro cultures IL-2 production was virtually undetectable in the in vitro activated In vitro cultured T cell lines were established by culturing 2 ϫ 107 spleen cell line after ionomycin treatment. This unresponsive state in- cells from transgenic mice in DMEM with 5% FCS and OVA at a final duced in the T cell lines was not due to decreased cell viability, as concentration of 1 mg/ml for 10 days. they were still able to proliferate when the treated cells were incubated with exogenous IL-2 (Fig. 1B). Naive, untreated T cells Induction of anergy with fixed APC that did not express significant amounts of the IL-2R, as expected, by guest on September 25, 2021 Preparation of fixed APC was performed under previously described con- did not proliferate in response to IL-2 exposure. However, naive T ditions (1). Briefly, BALB/c splenocytes were irradiated at a dose of 2600 cells treated with Ag and fixed APC did respond to IL-2 exposure rad and were incubated for1honicein0.44 ml of 0.9% NaCl containing (Fig. 1B). This indicates that the Ag presented by the fixed APC 75 nM EDCI (Calbiochem, La Jolla). The cells were washed extensively in serum-free medium to stop the coupling reaction. Normal APC consisted of induced a change in the naive cells, making them responsive to the BALB/c splenocytes irradiated at a dose of 2600 rad. T cells (2 ϫ 106 cells) cytokine. Thus, naive T cells appear to be fundamentally different were incubated with EDCI-treated or normal APC (10 ϫ 106 cells) in the from T cell clones in their responses to Ag presented by fixed APC ␮ presence of 50 M cOVA323–339 peptide in 24-well plates (Corning, Corn- and their susceptibility to become anergic by such means. ing, NY) in a total volume of 2 ml of DMEM with 5% FCS for 16 to 20 h. Cells were harvested and washed with medium three times, and 2 ϫ 104 recovered cells were stimulated with normal APC (5 ϫ 105) alone or with Differences in response to ionomycin treatments of naive T APC plus OVA (1 mg/ml) or PMA (10 ng/ml; Sigma Chemical Co., St. cells and T cell lines Louis, MO) and ionomycin (1 ␮M; Calbiochem, La Jolla, CA) in DMEM with 5% FCS a final volume of 200 ␮l in 96-well round-bottom plates Previous studies have demonstrated that anergy induction via ϩ (Costar, Cambridge, MA). After 24 h of culture, supernatants were har- chemically fixed APCs involves a rise in intracellular Ca2 (8). vested, and the presence of cytokine was measured. Furthermore, this anergic state can be reproduced by raising intracellular Ca2ϩ levels in susceptible T cell clones with ionophores Induction of anergy with ionomycin such as ionomycin. However, used in the context of additional 2ϩ Cells (1 ϫ 106) were incubated with ionomycin (Calbiochem) at a final stimuli, Ca influx can result in distinctly different responses in concentration of 1 ␮M for 20 h in DMEM with 5% FCS in a volume of 2 the cell. This is illustrated in Table I. In vitro cultured cell lines ml. Recovered cells were stimulated in the same manner as fixed APC- derived from TCR transgenic mice were stimulated with PMA, stimulated T cells for cytokine production. ionomycin, and the combination of PMA and ionomycin. Alone, PMA and ionomycin were insufficient to elicit significant IL-2 Proliferation and lymphokine assays production, while together they elicited a vigorous response. How- IL-2-dependent T cell proliferation was measured by cultured T cells (2 ϫ ever, ionomycin and PMA had drastically different effects on the 4 ␮ 10 ) in a final volume of 200 l of DMEM with 5% FCS containing IL-2 cell when the treated cells were subsequently challenged with Ag at a final concentration of 10 U/ml in 96-well flat-bottom plates (Costar). The cells were incubated at 37°C for 24 h and harvested following a 6-h and appropriate APC. The ionomycin-treated T cell line failed to incubation with tritiated thymidine (New England Nuclear, Boston, MA) at generate measurable IL-2 in response to Ag stimulation. PMA, in 1 ␮Ci/well. IL-2 activity was assayed using the IL-2-dependent CTLL line contrast, had no significant effect on the capacity of T cells to (23). IL-4 was measured using the IL-4-dependent cell line, 6I4, developed respond to the secondary stimulation. Thus, although both PMA in this laboratory. IL-3 was assayed using the IL-3-dependent line FDC-P3 (24). IFN-␥ activity was determined using the inhibition assay with the and ionomycin failed to elicit any IL-2 production from the T cells indicator line WEHI-279 (25). Units are defined as reciprocal dilution of when used alone, ionomycin, but not PMA, induced a unique culture supernatants that gave 50% of the maximum activity. change in the state of the cell to make it unresponsive to antigenic The Journal of Immunology 35

FIGURE 1. Differential effects of fixed APC plus Ag on naive and in vitro cultured T cells. Naive T lymphocytes isolated from TCR transgenic mice and T cell lines were exposed to medium alone (open circles) or with fixed APC and Ag (solid circles) as described in Materials and Methods. A, Recovered cells were stimulated with BALB/c APC with the indicated dose of antigenic peptide for 24 h. The cultures supernatants (final concentration, 50% of the original culture) were assayed for IL-2 activity. The data shown represent thymidine incorporation of the IL-2-dependent cell line, CTLL-2. B, IL-2 responsiveness of T cells exposed to fixed APC was measured by culturing T cells recovered after initial treatment with exogenous IL-2 (10 U/ml)

for 24 h. Thymidine incorporation per culture Downloaded from was measured as described in Materials and Methods. http://www.jimmunol.org/ Table I. Comparison of anergy induction between naive T cells and naive cell from that in the in vitro cultured T cell. Ionomycin was in vitro cultured T cell lines, with ionomycin treatmenta used in all subsequent experiments to further characterize the dif- ference between naive and activated T cells. This method is cell 1o IL-2 2o IL-2 density independent and thus excludes the possibility that the re- Cell Type Stimuli (U/ml) Stimuli (U/ml) sistance of the naive cells to the fixed APC treatments is due to Naive differences in engagement at the TCR level. The differences ob- None 0 None 0 served in this experimental system are more likely due to inherent Ova 60 differences in the T cell populations in their susceptibility to an- PMA 0 None 0 ergy induction. by guest on September 25, 2021 Ova 150 Ion 0 None 0 Ova 250 In vivo primed T cells are analogous to T cell lines in their susceptibility to anergy induction P ϩ I 5000 None ND Ova ND Most studies examining the effects of anergy-inducing stimuli have Cell line used T cell lines maintained in culture for at least several days. None 0 None 0 Since freshly isolated T cells are resistant to anergy induction, one Ova 30 explanation is that the maintenance of cells in culture renders them PMA 0 None 0 susceptible. Alternatively, if ionomycin sensitivity is a property Ova 15 acquired with T cell activation, primed T cells isolated directly Ion 0 None 0 from the immunized host should possess the same sensitivity to Ova 0 ionomycin as that observed in the T cell line. To address the sus- P ϩ I 750 None ND ceptibility of in vivo primed T cells to anergy induction, it was Ova ND necessary to acquire a means by which uniform cell populations a Freshly isolated naive T cells or in vitro-activated T cell lines from DO11.10 could be isolated from the host in both primed and unprimed states. mice were isolated and incubated in culture as a primary stimulation for 20 h Ideally, this would entail the identification of a cell population with ␮ with media alone (None); PMA, 10 ng/ml; ionomycin, 1 M (Ion); and PMA and a single TCR and would exclude those cells that may possess al- ionomycin together (P ϩ I). The cells were then washed three times with media and incubated with irradiated APC and media alone or 1 mg/ml OVA (Ova) as ternative receptors. To accommodate these needs, DO11.10 mice a secondary stimulation for 24 h. Supernatants from both primary and secondary were bred with mice who were homozygous for the disruption of cultures were assayed for IL-2 as described in Materials and Methods. Results are Ϫ/Ϫ expressed in U/ml. The above results are representative of three separate exper- the RAG-2 gene, (RAG-2 ) (22). This prevents the rearrange- iments. ND, not determined. ment of endogenous receptor genes and assures that all the T cells in the periphery possess a single, uniform TCR. These mice were used in the subsequent analysis. stimulation. This effect, however, was dependent upon the state of DO 11.10 TCR RAG-2Ϫ/Ϫ transgenic mice were immunized in the cell, as naive freshly isolated T cells maintained their ability to the footpad with OVA (100 ␮g) in CFA. The T cells from the produce IL-2 with Ag stimulation even after ionomycin treatment draining lymph nodes were isolated 7 days after immunization. (Table I). Naive cells, however, still appeared to be influenced by The in vivo primed T cell population was analyzed by surface ionomycin, as the magnitude of the secondary response was immunofluorescence and compared with cells from nonimmunized greater than that of the response of the untreated population. This mice. Lymph node cells from naive mice were stained with an indicates that ionomycin induces a distinctly different state in the anti-Id Ab (KJ1-26) and counterstained with an anti-CD62 Ab 36 IONOMYCIN-INDUCED ANERGY IN NAIVE AND ACTIVATED T CELLS

FIGURE 2. Surface CD62 expression in naive, in vivo activated, and in vitro activated T cells. Lymph node cells from nonimmunized TCR transgenic mice on a RAG-2-deﬁcient background (naive) and from Ag-primed DO 11.10 RAG-2Ϫ/Ϫ mice (primed lymph node) and in vitro cultured T cells from the same mouse strain (cell line) were stained with anti-idiotypic Ab to TCR (KJ1-26) and anti-CD62 Ab (MEL 14). Stained cells were analyzed by FACScan. The control consisted of lymph node cells stained with secondary reagents alone. The percentage of the total cells is indicated for Downloaded from each quadrant when more than one population exists. http://www.jimmunol.org/

Table II. Comparison of total cell number in the lymph node and Table III. Similar sensitivities of in vivo-primed and in vitro- spleen in naive and primed DO11.10 Rag-2Ϫ/Ϫ micea stimulated cell lines to ionomycin-induced anergya

Cell Number (ϫ10Ϫ6) Cell Type 1o Stimuli 2o Stimuli IL-2 (U/ml) by guest on September 25, 2021

Lymph Node Spleen Naive None None 0 P ϩ I 1,300 Naive 0.15 Ϯ 0.04 19 Ϯ 10 Ova 140 Primed 1.80 Ϯ 0.4 59 Ϯ 20 Ionomycin None 0 a Total cell numbers obtained from the spleen and lymph nodes of DO11.10 P ϩ I 1,400 Ϫ/Ϫ ␮ Rag-2 mice primed with 100 g of OVA in CFA in the footpad and harvested Ova 330 7 days later are compared with age-matched naive mice. Numbers reflect Ϯ mean SD of four mice in each group. Cell Line None None 0 P ϩ I 10,000 Ova 200 (MEL-14). As shown in Figure 2, the vast majority of TCR-pos- Ionomycin None 0 itive cells in the naive mouse express high levels of CD62 (L- P ϩ I 3,300 selectin). Upon priming with Ag, the number of cells in draining Ova 0 lymph node as well as in spleen increased significantly (Table II), Mel 14Ϫ None None 0 and the expansion of Ag-reactive T cells accounted for this in- Pma ϩ Ion 10,000 crease as the cells expressed the transgene-derived receptor, as Ova 660 measured by the Ab KJ1-26 (Fig. 2). A significant fraction of T Ionomycin None 0 cells from immunized mice were now low for CD62 expression, Pma ϩ Ion 1,400 consistent with the findings of other studies characterizing the sur- Ova 50 face phenotype of activated T cells (26, 27). a T cells from DO11.10 Rag-2Ϫ/Ϫ mice derived from naive T cells obtained from an unprimed animal (Naive), stimulation in vitro with Ag and APC for 7 To determine whether the in vivo activated population had ac- days (Cell Line), or KJ1-26 positive, MEL-14 low cells obtained by fluorescent quired sensitivity to ionomycin-induced anergy, the low CD62 cell sorting of LN cells primed in vivo with OVA in CFA for 7 days (Mel 14Ϫ) population was purified using FACS, so that Ͼ98% of the cells were each incubated with media alone (None), or ionomycin (1 ␮M) as the primary stimulus for 20 h. Each group was then washed and incubated with were low for expression of CD62 yet still expressed the transgene- media alone (None), PMA and ionomycin (P ϩ I), or OVA at 1 mg/ml with APC derived TCR. This purified T cell population was compared with (Ova) as a secondary stimulus for 24 h, and the supernatants were subsequently freshly isolated naive cells, which were stained with the same Abs assayed for IL-2 activity as outlined in Materials and Methods. The above results are representative of three separate experiments. as the in vivo primed cells, and the in vitro cultured T cell line, all derived from the DO11.10 TCR RAG-2Ϫ/Ϫ mouse line for their sensitivity to ionomycin. The three cell populations were incubated tured T cell line was rendered unresponsive with ionomycin treat- with ionomycin for 16 to 20 h, washed, and rechallenged with Ag ment, while the naive T cell remained reactive after the same treat- and APC. Consistent with the previous studies, the in vitro cul- ment (Table III). These cells were still viable and capable of IL-2 The Journal of Immunology 37

Table IV. Ionomycin’s anergy induction primarily affects IL-2 tion. In vitro cultured T cells can be rendered anergic by either Ag expression in in vitro-cultured T cell lines with little influence on presented by fixed APC or by exposure to ionomycin. These T a other cytokines cells fail to produce IL-2 or proliferate upon rechallenge with Ag presented by functional APC. The treated cells are still viable, as b Cytokine Assay they proliferate in response to exogenous IL-2 and produce IL-2 in 1o Stimuli 2o Stimuli IL-2 IL-3 IL-4 IFN-␥ response to PMA and ionomycin. In contrast, naive T cells are resistant to such anergy induction and maintain their ability to None None 0 0 0 0 generate IL-2. This resistance is not due to a lack of signaling of P ϩ I 5000 660 33 140 Ova 100 2000 10 33 the T cells with these stimuli, since naive T cells exposed to such stimuli exhibit an increased reactivity to IL-2 and greater cytokine- Ion None 0 0 0 0 producing capacity than nontreated naive T cells. This functional P ϩ I 500 2000 142 110 Ova 0 660 33 5 change indicates that events elicited in the naive cell by the anergy-inducing stimuli are distinct from those induced by in vitro a In vitro-cultured T cell lines from DO11.10 Rag-2Ϫ/Ϫ were incubated with media alone, (None) or ionomycin (Ion) as a primary stimulus for 20 h, then activated T cells. washed and incubated with media alone (None), PMA and ionomycin (P ϩ I), or The data presented above indicate that naive cells can be con- OVA plus APC (Ova) as a secondary stimulus for 24 h and assayed for cytokine verted into ionomycin-susceptible cells with conventional immu- activity. b Results from cytokine assays for IL-2, IL-3, IL-4, and IFN-␥ are expressed in nization. This was demonstrated in TCR transgenic mice on a Ϫ Ϫ U/ml. The above results are representative of three separate experiments. RAG-2 / background in which all the T cells expressed only one Downloaded from receptor. After priming, this uniform T cell population expanded in significant numbers in response to the Ag. A large fraction of cells expressed low levels of CD62 (L-selectin), but a significant num- production upon stimulation with PMA and ionomycin. Thus, the ber of T cells remained high in CD62 expression. Since the low unresponsive state elicited by ionomycin was not due a toxic effect CD62 population alone cannot account for the entire increase in on the cells. When the in vivo primed, low CD62 T cells were the number of T cells, some T cells were stimulated by Ag and exposed to ionomycin and tested for their Ag reactivity, their ca- http://www.jimmunol.org/ maintained high CD62 expression. We observed similar heteroge- pacity to produce IL-2 in response to Ag was greatly reduced com- neity for IL-2R and CD44 ( pgp-1) expression (data not shown). It pared with the that of the same T cell population not exposed to is not clear at present whether this heterogeneity represents a dis- ionomycin. This experiment was performed three times, and in tinct T cell activation state. However, it is likely that this hetero- each case, ionomycin treatments of the in vivo primed, low CD62 geneity represents different stages of the T cell as it undergoes population reduced the amount of IL-2 production in response to Ag-driven proliferation. Alternatively, the increase in the total Ag stimulation by at least 1 order of magnitude. This effect was number of high CD62 cells could be due to the migration of T cells simply not due to the effects of the Abs used to isolate the cells, into the lymphoid tissues that have not yet encountered Ag and since similar treatments of the naive population failed to affect

lowered their expression of CD62. Thus, to restrict our analysis to by guest on September 25, 2021 their resistance to ionomycin. Thus, in vivo activated T cells are an Ag-primed population, we have used exclusively low CD62 T sensitive to ionomycin-induced anergy induction in a fashion anal- cells and have demonstrated that the conversion from a naive to a ogous to that of the in vitro activated T cell line. primed T cell renders the cell susceptible to ionomycin-induced The susceptibility to anergy induction is confined to IL-2 anergy. The relationship among surface phenotypic changes, cell expression cycle regulation, and susceptibility to anergy induction in primed mice requires further investigation to help elucidate a biochemical Further analysis was performed to determine whether other cyto- basis for the anergy susceptibility. kine production pathways were affected by ionomycin in the same Other investigators have noted previously that naive cells ap- fashion as IL-2. In vitro cultured T cells were incubated with iono- peared to be uninfluenced by the effects of fixed APC treatments mycin for 16 h and stimulated with Ag and APC. Culture super- (28). These studies were unable to clearly demonstrate differences natants were tested for IL-2, IL-3, IL-4, and IFN-␥. Similar to the between primed and naive cells in IL-2 production following ex- previous assay, ionomycin-treated cells produced no IL-2 upon posure to fixed APCs and concluded that the means by which restimulation with Ag and APC. However, IL-4 production re- anergy is induced may only affect certain T cell subtypes. Our mained comparable to that of untreated T cells. In contrast, the assay systems, however, were able to detect clear differences be- amounts of IFN-␥ and IL-3 produced by ionomycin-treated cells tween naive and primed T cells in their susceptibility to anergy were significantly lower than those produced by nontreated cells, induction. By using cell sorting for a low CD62 population to but were still readily detectable (Table IV). This experiment was isolate in vivo primed T cells and by allowing the in vitro activated performed three times. In each instance, IL-4 production after Ag cell line to incubate for a sufficient amount of time in culture, we stimulation did not decrease in response to ionomycin treatment, were able to analyze more uniform populations that were devoid of while the effects of ionomycin on IFN-␥ and IL-3 expression failed cells that may be at different stages of activation and thus may to reduce the response more than 3- to 10-fold, with detectable possess different susceptibilities to anergic stimuli. In addition, the activity remaining. This is in contrast to IL-2 production, for which use of ionomycin as the means of inducing anergy ensured that the ionomycin treatments resulted in no measurable biologic activity T cells assayed were uniformly treated so that the response as- in the in vitro cell line after Ag stimulation. Thus, each of these sessed was not dependent upon the degree of engagement of the cytokines must be regulated in a different fashion than IL-2, and cell’s receptor with a fixed APC, which may fail to induce anergy. anergy induction seems to be specific for IL-2 and IL-2-dependent Our data suggest that selecting for cells expressing low CD62 ex- proliferation of T cells in vitro. pression is a means of identifying a T cell population that is sensitive to the effects of anergic stimuli. Discussion Ionomycin treatments have different effects on different cytokine We have demonstrated that the naive T cells are distinctly different production pathways. IL-2 production is dramatically reduced with from preactivated T cells in their susceptibility to anergy induc- subsequent Ag stimulation, while IL-4, in contrast, appears to be 38 IONOMYCIN-INDUCED ANERGY IN NAIVE AND ACTIVATED T CELLS unaffected. Ionomycin reduced the responses of IFN-␥ and IL-3 8. Jenkins, M. K., D. M. Pardoll, J. Mizuguchi, T. M. Chused, and R. H. Schwartz. production to Ag, but its effects on these two cytokines were in- 1987. Molecular events in the induction of a nonresponsive state in interleukin 2-producing helper T-lymphocyte clones. Proc. Natl. Acad. Sci. USA 84:5409. complete. The T cell lines used in these experiments were only 9. Cho, E. A., M. P. Riley, A. L. Sillman, and H. Quill. 1993. Altered protein stimulated for 7 days and were thus heterogeneous. Cells within tyrosine phosphorylation in anergic Th1 cells. J. Immunol. 151:20. this mixture predominantly consist of CD4 Th cells expressing 10. Sloan-Lancaster, J., A. S. Shaw, J. B. Rothbard, and P. 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